Selected genes with both average difference and two-consecutive differences were further validated with Sequenom Mass ARRAY. Epityper DNA methylation analysis is based on bisulfite conversion of DNA, PCR amplification, followed by in vitro transcription and analysis of cleaved products by Mass Spectrometry (MALDI-TOF). The primers were designed using MethPrimer (www.urogene.org/methprimer) for 26 gene promoter regions with 48 amplicons (103-625bp with median target length of 400bp) covering, on average, 9 CpGs per amplicon. In brief, DNA was isolated as described above for MeDIP analysis from differentiated DRG cells with or without alcohol treatment (n=1 each). The methylation detection was carried out in the Sequenom facility in San Diego, CA. Approximately 1 μg of DNA from each sample was treated with sodium bisulfite using the EZ DNA methylation kit (ZymoResearch, CA) according to the manufacturer's protocol, and amplification was done using the primers with the T7 promoter tag on the reverse primer. The in vitro transcription was done, followed by site-specific cleavage using MassCLEAVE biochemistry. MassARRAY compact MALDI-TOF mass was used to acquire spectral peaks and converted into methylation ratios using EpiTYPER
